Current Biology, Volume
24
Supplemental Information
Anaerobic Bacteria Grow within
Candida albicans
Biofilms and Induce
Biofilm Formation in Suspension Cultures
Emily P. Fox,
Elise S. Cowley,
Clarissa J. Nobile,
Nairi Hartooni,
Dianne K. Newman,
and Alexander D. Johnson
!
!
!
Figure!S1,!Related!to!Figure!1.!Bacteria!are!incorporated!throughout!the!
biofilm.!
C.#albicans#
was!grown!in!biofilms!for!24!hours!either!alone!(A),!or!with!
E.#
!
coli#
(B),!
K.#pneumoniae#
(C),!
E.#faecalis#
(D),!
C.#perfringens
!
(E),!or!
B.#fragilis#
(F).
!
Biofilms!were!stained!with!conconavalin!A!
–
!
Alexa!594!and!Syto!13!dyes,!then!
imaged!by!CSLM.!Images!shown!are!top!vie
w,!maximum!intensity!projection!z
O
stacks!of!65
!
μm
!
thick!sections!internal!to!the!biofilm.
!
Scale!bars!are!50!μm.!G)!
Biofilm!thickness!of!at!
least!
six
!
technical!and!
two
!
biological!replicates!was!
measured!using!CSLM.!Average!values!shown,!error!bar
s
!
indicate!standard!
deviation.!*Significantly!different!from!
C.#albicans#
alone
,!
P
!
<!0.001,!by!student
’
s!
paired!t
O
test.!
!
!
!
!
!
!
!
Figure!S2,!related!to!F
i
gure!2.!
Incorporation!of!
C.#albicans#
and!bacteria!in!
biofilms!is!affected!by!co
>
culture.!
Measurement!of!
cfu
/ml!of!indicated!species!
grown!in!biofilms!in!monoculture!or!co
O
culture,!in!biofilms!in!ambient!oxic!
conditions.!A)!
C.#albicans
#
SN250
!
and/or!
E.#
coli
.!B)!
C.#albicans
!
SN250!
and/or!
K.#
pneumoniae
.!C)!
C.#albicans
!
SN250!
and/or!
E.#faecalis
.!D)!
C.#albicans#
P57055!and/or!
C.#
perfringens
!
E)!
C.#albicans#
CEC3494!and/or!
C.#perfringens
.!
S
ho
wn!is!the!mean!of!at!
least!two
!
replicates,!error!bars!are!standard!deviat
ion.
!
!
!
!
!
!
Figure
!
S3,!Related!to!Figure!3
.!
WOR1#
is!regulated!independently!from!other!
opaque!regulators!during!biofilm!co
>
culture
.!
A)!Confocal!Scanning!Laser!
Microscopy!of!wild!type
!
(SN250)
!
or!
wor1∆/∆
!
(TF176)!
strain!co
O
cultured!with!or!
!
without!
K.#pneumoniae#
(abbrevia
ted!
Kp).!Images!shown!are!maximum!intensity!
projections!of!the!top!and!side!view.!!Scale!bars!are!50!μm.!B)!Top!
two!
rows:!heat!
map!of!gene!expression!in!
C.#albicans
!
(SN250)!
when!co
O
cultured!with!
K.#pneumoniae#
in!biofilms,!compared!to
!
C.#albicans#
alone.!Values!are!the!median!values!of!at!least!
two!biological!replicates.!!Bottom
!
two
!
rows:!heat!map!of!gene!expression!in!opaque
!
(AHY136)
!
compared!to!white!cells
!
(AHY135)
;!and!genes!whose!promoters!are!
bound!by!Wor1
,!data!from
!
[
S
1]
.!
A
long!the!
x
O
axis
!
are!6,111!genes
.!
Yellow!genes!are!
upregulated!or!bound!by!Wor1,!Blue!genes!are!downregulated,!gray!genes!are!not!
bound!by!Wor1.!
C
O
D)!Quantitative!RT
O
PCR.!
Shown!are!the!mean!values!
of!at!least!
two!biological!replicates.!Error!bars!are!standard!deviation.
!
WT!=!SN250,!Kp!=!
K.#
pneumoniae
.!C
)!
Expression
!
of!
WOR1
!
in!the!indicated!strains
.
#
!
D
)!
Expression
!
of!
WOR1,#WOR2,#CZF1,#
and!
PTH2#
in!the!indicated!strains
.
!
!
!
!
!
Figure!
S4,!Related!to!
Figure!4
.!Characterization!of!
C.#perfringens
>
induced!
aggregates
!
when!co
>
cultured!in!ambient!oxic!cultures!with!
C.#albicans
.!
!
A)!
Heat!
map!of!gene!expression!in!
C.#albicans
!
when!co
O
cultured!in!suspension!with!
C.#
!
perfringens,#
compared!to!
C.#albicans
!
alone.!Sh
own!are!genes!differentially!regulated!
more!than!
two
!
fold.!Values!are!the!median!values!of!at!least!two!biological!
replicates.!Upregulated!genes!are!yellow,!downregulated!genes!are!blue.!*Genes!that!
are!regulated!during!hypoxia!in!
C.#albicans;#
these!genes!
are!significantly!enriched!in!
our!dataset!with!a!p!<1.5X10
O
5!
by!the!chi
O
squared!test
.!B)!
C.#albicans#
was!grown!with!
or!without!
C.#perfringens
,!in!biofilms!for!1!hour.!Biofilms!were!stained!with!
conconavalin!A!
–
!
Alexa!594!and!Syto!13!dyes,!then!imaged!by!CS
LM.!Images!shown!
are!maximum!intensity!projections!of!the!top!view.!Scale!bars!are!50!μm.
!
At!least!
two!replicates!were!visualized,!representative!images!are!shown.!
C
!
O
!
E
)!Suspension!
cultures!of!the!indicated!
C.#albicans#
strains,!with!or!without!
C.#perfring
ens
,!grown!for!
4!hours!at!37°C.!Assay!was!performed!at!least!twice!for!each!condition!or!mutant!
strain.
!
C)!
C.#albicans#
wild!type!or!
indicated!
mutant!strains
.!D)!
C.#albicans#
complemented!strains!where!a!wild!type!copy!of!indicated!gene!is!restored!in!the!
deletion!strain!background.!E)!Aggregation!was!measured!using!
an!adapted!assay!
from!
S.#cerevisiae,#
in!which!flocculation!is!associated!with!greater!sedimentation!of!
aggregates
,!as!measured!by!optical!density!(OD
600
)
.#
Assay!was!performed!at!least!
twice,!error!bars!are!standard!deviation.!
Cp#
=
#
C.#perfringens.#
*Significantly!different!
from!
WT#+Cp
,!P
!
<
!
0.05.!#Significantly!different!from!
WT#
alone,!P
!
<
!
0.05.
!
!
**Significantly!differe
nt!between!indicated!deletion!strain!and!complemented!strain,!
P
!
<
!
0.05.!
S
tudent
’
s!paired!t
O
test
!
was!used!to!calculate!significance.
!
!
!
!
!
!
Supplementa
l
!
Text
!
!
Wor1!is!upregulated!independently!from!other!opaque
O
enriched!transcription!
regulators
!
during!biofilm!co
O
culture
!
!
To!explore!the!role!of!
WOR1
!
in!co
O
culture,!we!used!a!
wor1∆/∆#
strain!in!biofilm!co
O
cultures!with!
K.#pneumoniae.#
We!found!no!
morphological
!
perturbations!when!
biofilms!were!viewed!by!CSLM,!and!gene!expression!microarrays!
revealed!few!
changes!in!the!transcriptional!profile!induced!by!co
O
culture!with!
K.#pneumoniae#
(Figure!S3A,!B)
.
!
It
!
is!known!that!in!opaque!cells
,
!
Wor1!is!part!of!a!closely!
intertwined!transcriptional!regulation!network,!where!it!regulates!and!is!regulated!
by
!
several!other!regulators.!Both!co
O
culture!with!
K.#pneumoniae
!
and!switching!from!
white!to!opaque!increases!expression!of!regulators!
WOR1,#
WOR2,#WOR3,#
and!
CZF1
,#
and!so!we!examined!the!connections!between!
WOR1#
expression
#
and
!
the!expression!
of
!
other!white
O
opa
que!regulators
#
in!co
O
culture!with!
K.#pneumoniae
.
#
We!used!RT
O
qPCR!and!the!tiling!probes!in!our!arrays!that!cover!
WOR2,#WOR3,#
and!
CZF1
!
to!
determine!whether!expression!of!any!of!these!regulators!depend!on!Wor1,!and!
whether!expression!of!
WOR1
!
depend
s
!
on!any!of!these!regulators,!
and
!
found!that!
they!are!independently!induced!(Figure!S
3C
,!Dataset!1
,!
2
).!
WOR1#
expression!is!also!
independent!of!Wor2,!Wor3,!and!Czf1!in!white!and!opaque!cells
,
!
and!it!is!impossible!
to!assay!the!effect!of!
WOR1
!
deletion!in!opaqu
e!cells!on!expression!of!the!other!
regulators,!because!
wor1∆/∆#
cells!are!locked!in!the!white!state!
[
S
1]
.!!Therefore!the!
independent!nature!of!the!induction!of!these!regulators!in!co
O
culture!does!not!
necessarily!distingu
ish!it!from!the!regulatory!circuitry!used!in!the!white
O
opaque!
switch.
!
We!found!that!expression!of!
PTH2
!
was!induced!by!co
O
culture!with!
K.#
pneumoniae
#
(Figure!3B)
,!which!is!interesting!because!
PTH2#
is!a!homolog!of!
WOR1
,!
and!has!the!same!DNA!binding!domain,!bu
t!the!deletion!strain!has!no!known!
phenotype!and!no!previously!identified
!
condition!induces!its!expression.!
Deletion!of!
!
WOR1
!
did!not!affect!
PTH2#
expression,!and!deletion!of!
PTH2#
did!not!affect!
WOR1#
expression,!so!we!constructed!a!
wor1∆/∆pth2∆/∆#
double!dele
tion!mutant,!and!
found
#
that!deletion!of!
WOR1#
and!
PTH2
!
had!no!effect!on!expression!of!
WOR2#
or!
CZF1#
(Figure!S
3D
).!!We!have!found!a!condition!in!which!
PTH2#
is!expressed,!but!it!remains!
to!be!determined!what!role!
Pth2
!
plays!during!co
O
culture.
!
!
!
C.#albicans
!
expresses!genes!
associated!with
!
hypoxia
!
during!suspension!co
O
culture!
with!
C.#perfringens
#
!
We!used!microarrays!to!measure!gene!expression!in!
C.#albicans
!
grown!in!ambient!
oxic!suspension!culture,!with!and!without!
C.#perfringens#
(Figure!
S4
A!and!Dataset!
3
)
.
!
Very!few!genes!were!differentially!regulated!during!planktonic!co
O
culture!with!
C.#
perfringens.
!
Using!a!four
O
fold!cutoff,!only!two!genes!were!upregulated:!
ORF
19.2800
!
and!
ORF
19.4763
,!both!of!which!encode!proteins!of!unknown!functions.!Using!a!two
O
fold!cutof
f,!ten!genes!were!upregulated!and!nine!genes!were!downregulated.!The!
majority!of!the!upregulated!genes!are!uncharacterized!and!the!downregulated!genes!
span!a!variety!of!functions,!including!the!genes!encoding!an!alternative!oxidase,!
Aox2,!a!surface!antigen
,!Csa1,!and!genes!encoding!for!proteins!involved!in!sugar!
acquisition,!Hgt1!and!Gal7.!Interestingly,!the!gene!set!differentially!regulated!in!
suspension!co
O
cultures!is!significantly!enriched!for!genes!regulated!during!hypoxic!
conditions!(p!<!1.5X10
O
5
)!
[
S
2]
,!
indicating
!
hypoxia!may!
occur
!
during
!
aggrega
tion,!
which!could!explain!the!increased!survival!of!
C.#perfringens
!
when!grown!aerobically!
with!
C.#albicans
.
!
!
!
!
!
Supplemental!Tables
!
!
Table!S1,!Related!to!Figure!4.
!
Induction!of!
C.#albicans
!
aggregation
!
by!bacteria!
during!suspension!co
O
culture!in!oxic!or!anox
ic!conditions
!
Strain
'
Ox
ic,'4
'
hours
'
Anox
ic,'4
'
hours
'
Oxic
,'24
'
hours
'
Anox
ic,'24
'
hours
'
C.#albicans#
alone
#
cloudy
'
cloudy
'
cloudy
'
cloudy
'
C.#perfringens#
alone
#
clear
'
cloudy
'
clear
'
cloudy
'
B.#fragilis#
alone
#
clear
'
clear
'
clear
'
cloudy
'
E.#coli#alone
#
cloudy
'
cloudy
'
cloudy
'
cloudy
'
E.#faecalis
'
alone
#
cloudy
'
cloudy
'
cloudy
'
cloudy
'
K.#pneumoniae
'
alone
#
cloudy
'
cloudy
'
cloudy
'
cloudy
'
C.#albicans#+#C.#perfringens
#
clumps
'
cloudy
'
cloudy/clumps
'
cloudy
'
C.#albicans#+#B.#fragilis
#
cloudy/clumps
'
cloudy
'
cloudy
'
cloudy
'
C.#albicans
#
+#E.#coli
#
cloudy/clumps
'
cloudy
'
cloudy/clumps
'
cloudy
'
C.#albicans#+#E.#faecalis
#
cloudy/clumps
'
cloudy
'
cloudy/clumps
'
cloudy
'
C.#albicans#+#K.#pneumoniae
#
cloudy
'
cloudy
'
cloudy
'
cloudy
'
!
!
Table!S
2
,!Related!to!Figure!
4
.
!
Screen!of!
C.#albicans
!
deletion!mutant!strains!for!
aggregation
!
during!
oxic!suspension
!
co
O
culture!with!
C.#perfringens
.#
(See!spreadsheet!
for!Table!S
2
).
!
!
Table!S
3
,!Related!to!Experimental!Procedures.!
Strains.!
(See!spreadsheet!for!
Table!S
3
)
!
[
S
3
–
S
1
3]
.
!
!
Table!S
4
,!Related!to!Experimental!Procedures.!
Primers.!
(See!spreadsheet!for!
Table!S4).
!
!
!
!
!
Supplementa
l
!
Experimental!Procedures
!
!
Culture!growth!and!maintenance
!
!
C.#albicans
!
was!streaked!from!a!glycerol!stock!onto!Yeast!Peptone!Dextrose!(YPD)!
agar!plates!and!grown!at!room!temperature!or!30
°
C.!Suspension!cultures!were!
grown!in!YPD!media!at!30
°
C,!with!aeration.!
C.#perfringens
!
and!
B.#fragilis
!
glycerol!
stocks!were!streaked!on!blo
od!agar!plates!and!grown!at!37
°
C,!in!an!anaerobic!jar.!
Suspension!cultures!were!grown!statically!in!Brain!Heart!Infusion!(BHI)!medium,!
supplemented!with!5%!fetal!bovine!serum!(BHI
O
FBS),!at!37
°
C,!in!an!anaerobic!jar.!
K.#
pneumoniae,#E.#faecalis,#
and
#
E.#coli
!
glycerol!stocks!were!streaked!on!
Luria!Broth!(
LB
)
!
agar!plates!and!grown!at!37
°
C!in!a
mbient!air
.!Suspension!cultures!were!grown!in!
BHI
O
FBS,!shaking,!at!37
°
C.
!
!
Extended!
b
iofilm!
a
ssay
!
!
A!6
O
well!polystyrene!plate!was!incubated!overnight!with!4!ml!bovine!serum!
per!
well.!Bovine!serum!was!removed,!wells!were!washed!with!4!ml!PBS,!and!4!ml!of!
BHI
O
FBS!medium!was!added.!Media!was!buffered!with!67!mM!K
2
HPO
4!
and!33!
m
M!
KH
2
PO
4
!
for!
K.#pneumoniae,
!
E.#faecalis#
and!
E.#coli
!
cultures!to!prevent!changes!in!pH.!
C.#
perfringens#
an
d!
B.#fragilis#
do!not!change!the!pH!of!the!culture!and!do!not!grow!well!
in!the!buffered!media,!so!the!buffer!was!omitted!for!these!species.!
C.#albicans
!
monocultures!were!buffered!or!unbuffered!to!match!co
O
cultures!with!each!species.!
Overnight!cultures!of!
C.
#
albicans
!
and/or!bacterial!cells!were!each!inoculated!to!
1X10
7
!
cfu
/ml.!Cells!were!adhered!at!37
°
C,!200!rpm
!
in!an!ELMI!shaker,!for!90!min.!
Unadhered!cells!were!washed!off!with!4!ml!PBS,!and!fresh!4!ml!BHI
O
FBS!was!added.!
Biofilms!were!allowed!to!form!for!4!or!24!
h
,!at!37
°
C,!200!rpm!in!an!ELMI!shaker,!in!
either!
ox
ic!or!an
ox
ic!conditions.
!
!
Extended!
s
uspension!
c
o
O
cu
ltures
!
!
!
Overnight!cultures!of!each!species!were!inoculated!to!1X10
6!
cfu
/ml!in!2ml!BHI
O
FBS!
media!or!phosphate!buffered!BHI
O
FBS!media!(see!description!in!Biofilm!Assay,!
above).!Cultures!were!shaken!vigorously!(225!rpm!in!an!orbital!shaker)!for!4!h!or!
24!h
!
at!37
°
C,!either!in!oxic!or!anoxic!conditions.!Cultures!were!screened!visually!for!
aggregation!after!4!h!of!incubation.!
!
!
Confocal!Scanning!Laser!Microscopy!(CSLM)
!
!
Biofilms!were!
dyed
!
with!50!μg/ml!Conc
a
navalin!A!
–
!
Alexa!594!conjugate!(Life!
Technologies!
C11253)
!
and!2!μM!Syto!13!nucleic!acid!stain!(Life!Technologies!
S7575)!for!1!
h
,!37
°
C,!200!rpm
!
in!an!ELMI!shaker,!in!the!dark.!Medium!containing!the!
dye
!
was!removed!and!biofilms!were!imaged!as!described!previously!
[
S
6]
.!!
!
!
Extended!
c
olony!
f
orming!
u
nit
!
(
cfu
)!
a
ssay
!
!
C.#albicans
,!and!each!bacterial!species!were!grown!in!biofilms!or!in!suspension!
cultures,!alone!or!pairwise!with!yeast!and!bacteria.!Cultures!were!grown!under!
ox
ic!
(ambient!air)!or!an
ox
ic!(inside!an!anaerobic!jar!with!
a!
GasPak,!BD!260678)!
conditions.!Cells!from!biofi
lms!were!collected!at!90!min!(immediately!after!
adherence),!4!
h
,!or!at!24!
h
!
by!removing!media,!washing!biofilms!with!4!ml!PBS,!then!
collecting!cells!in!4!ml!PBS,!using!a!cell!scraper!and!transfer!pipet.!Cells!were!
vortexed!vigorously!for!1!min!to!break!up!
clumps,!and!serial!dilutions!were!
performed.!For!suspension!cultures,!serial!dilutions!were!performed!directly!from!
cultures.!Dilutions!were!plated!onto!YPD!agar,!
in!ambient!air
,!at!30
°
C!to!count!
C.#
albicans#
cfu
.##
Dilutions!were!plated!on!blood!agar,
!
i
n!an!anaerobic!jar
,!at!37
°
C!to!
count!
C.#perfringens#
and!
B.#fragilis#
cfu
.
!
Dilutions!were!plated!on!LB,!
in!ambient!air
,!at!
37
°
C!to!count!
E.#faecalis,#E.#coli,#
or!
K.#pneumoniae#
cfu
.#
The!initial!inoculated!media!
for!both!biofilms!and!suspension!culture
s!were!also!plated!to!determine!the!starting!
cfu
/ml.!Biofilms!were!seeded!at!1X10
7
!
cfu
/ml!and!suspension!cultures!were!seeded!
!
at!1X10
6
!
cfu
/ml.!At!least!two!biological!replicates!and!three!technical!replicates!
were!performed!for!each!sample.
!
!
Oxygen!
m
easurements
!
!
Biofilms!used!for!oxygen!measurements!were!grown!as!described,!but!on!bovine!
serum!coated!latex!sheets!stretched!over!a!ring!of!polyvinyl!chloride!(PVC)!tubing.!
We!measured!
cfu
/ml!to!confirm!that!this!substrate!produces!biofilms!ca
pable!of!
supporting!
C.#perfringens
!
and!
B.#fragilis
!
and!found!it!just!as!effective!as!using!
polystyrene!plates!as!a!substrate.!We!used!this!set!up!so!that!latex!could!be!laid!atop!
an!agar!plate!prior!to!oxygen!measurement
s
,!in!order!to!protect!the!tip!of!
the!
oxygen!sensor!probe!as!it!passes!from!biofilm!to!substrate.!Oxygen!concentration!
was!measured!as!in
!
[
S
14]
,!
but!with!the!following!modifications.!We!used!a!Unisense!
STOX
O
Sensor!microelectrode!that!is!based!on!the!Clark
O
type!oxygen!sensor,!but!ha
s!
two!cathodes!to!amplify!the!oxygen!signal
,
!
allow
ing
!
detection!of!trace!levels!of!
oxygen!down!to!10!nM!
[
S
15]
.!
The!sensor
!
electrode!was!polarized!with!
O
0.8!V!after!
connection!to!a!picoampere!amplifier!on!a!multimeter.
!
!
Readings!were!acquired!
using!SensorTrace!pro!3.1.3!software.!The!calibration!curve!was!a!two!point!curve!
with!a!zero!point!and!the!fully!saturated!solution!be
ing!271.4!
μ
M,!using!the!
temperature!25
°
C!and!a!salinity!of!0.75%.!Atmospheric!oxygen!was!measured!in!a!
calibration!chamber!containing!water!bubbled!with!air!at!22°C.!
The!z
ero!reading!
was!obtained!by!measuring!a!solution!of!sodium!hydroxide!and!sodium!ascor
bate!at!
22°C.!To!measure!biofilms,!the!sensor!probe!was!positioned!above!the!biofilm,!using!
a!Leica!MZ!9.5!stereomicroscope!to!confirm!position;!readings!were!taken!every!10!
μ
m,!for!3!seconds.!The!biofilms!were!covered!in!PBS!during!the!measurements.!At!
le
ast!two!technical!replicates!were!performed!for!each!sample.!Three!successive!
replicate!profiles!were!taken!in!each!position.
!
!
!
RNA!extraction
!
!
!
C.#albicans
!
biofilms!were!grown!with!or!without!bacteria,!as!described!above,!in!6
O
well!plates!for!24!h.!Media!w
as!removed,!biofilms!were!gently!washed!with!4!ml!
PBS,!and!biofilms!were!collected!in!fresh!4!ml!PBS!by!scraping!with!a!cell!scraper!
and!transferring!to!a!50!ml!conical!tube.!6!wells!were!pooled!from!each!plate!
containing!one!condition.!Biofilms!samples!we
re!centrifuged!at!3,000!X!g!for!5!min!
and!supernatant!discarded.!Biofilms!were!stored!at!
O
80
°
C.!RNA!was!extracted!as!
previously!described!
[
S
16]
,!using!the!RiboPure!
–
!
Yeast!RNA!kit!(Ambion
,!AM1926),!
following!the!manufacturer’s!protocol.
!
!
Gene!
e
xpression!
m
icroarrays
!
!
Gene!expression!microarrays!were!performed!as!previously!described!
[
S
17
]
.!Briefly,!
cDNA!was!synthesized!from!8!
μ
g!total!RNA!using!Superscript!II!Reverse!
Transcriptase!(Invitrogen,!
18064
O
014),!according!to!the!manufacturer’s!instruction,!
and!using!a!mixture!of!0.5!mM!3:2!aminoally
l
O
dUTP!(Ambion!8437):dNTPs.!cDNA!
was!dye!cou
pled!to!either!Cy3!or!Cy5!(GE!Healthcare!PA23001!or!PA25001),!then!
0.4!
μ
g!Cy3
O
labelled!cDNA!and!0.4!
μ
g!Cy5
O
labelled!cDNA!were!hybridized!onto!
custom!Agilent!8
X
15k!microarrays!(AMADID!#020166),!contain
ing
!
at!least!two!
independent!probes!for!each!ORF.!Slides!were!scanned!on!a!4000B!Axon!Instrument!
Scanner.!Data!was!analyzed!and!visualized!as!described!previously!
[
S
6]
!
using!
GenePix!Pro!software!version!7,!LOWESS!normalization,!Cluster!version!3.0,!and!
Java!TreeView!version!1.1.6r2.!At!least!
two
!
biological!replicates!were!performed!for!
each!condition.!We!also!performed!arrays!comparing!
C.
#
albicans#
+
E.#coli#
K12!type!
strain!to!
C.#albicans#
alone,!and!found!they!were!very!similar!to!the!arrays!performed!
on!samples!containing!the!clinical!
E.#coli#
strain!used!throughout!this!study!(Dataset!
1
).!Gene!expression!microarray!data!are!reported!in!Data
set!1
!
(biofilm!co
O
culture),!
Dataset!
2
!
(
wor1∆/∆#
co
O
culture,!additional!probes),!and!
Dataset!
3!(suspension!co
O
culture).!Raw!gene!expression!array!data!
was!uploaded!to!
the!Gene!Expression!
Omnibus
!
database
!
(
www.ncbi.nlm.nih.gov/geo
;
!
accession!#
GSE55026)
!
!
!
Strain!
c
onstruction
!
!
Deletions!strains!that!were!screened!in!the!aggregation!assay!are!listed!in!Table!
S2
,!
including!the!full!transcription!factor!deletion!library!and!several!additional!
mutants!published!previously!
[
S
4]
.!Table!
S2
!
also!indicates!which!strains!were!
confirmed!for!phenotype!by!testing!a!second,!independent!deletion!mutant!isolate!
(
8
/1
1
!
strains)
!
and/or!testing!a!strain!complemented!with!a!wild!type!copy!of!the!
deleted!gene!(
9
/1
1
!
strains).!
!
All!
11
!
mutant!phenotypes!were!confirmed!with!at!least!
one!of!these!methods.
!
C.#albicans
!
strains!with!phenotypes!in!the!screen!or!strains!
that!are!used!in!other
!
assays!in!this!study
,!as!well!as!bacterial!strains,
!
can!be!found!
in!Table!
S3
.
!
All!primer!sequences!used!in!this!study!are!listed!in!Table!
S
4
.!
Homozygous!gene!deletion!mutant!strains!and!
HIS1
!
or!
LEU2
!
addback!strains!were!
constructed!as!in!
[
S
3]
,!using!primers!A!and!B!to!amplify!th
e!5’!flank!of!the!targeted!
ORF,!primers!C!and!D!to!amplify!the!3’!flank.!Integration!was!checked!using!primer!
E!with!RZO39!or!41!and!primer!F!with!RZO40!or!42!and!the!ORF!was!detected!with!
primers!G!and!H.!Some!strains!were!signature!tagged!with!a!unique!2
0
O
mer!
sequence,!using!the!indicated!primers.!TF183,!was!made!with!pSFS2a
O
CPH1KO,!as!
described!previously!
[
S
18]
.!The!
wor1∆/∆pth2∆/∆#
strain!was!constructed!by!two!
rounds!of!
PTH2
!
gene!replacement!in!the!
wor1∆/∆#
background!strain!(TF176),!using!
the!recycla
ble!SAT1/flipper!cassette!in!plasmid!pSFS2A,!described!previously!
[
S
19]
.!
!
BRG1#
and!
RIM101
#
complementation
!
strains!
EF93!and!EF95!were!made!by!
integration!of!pEF3!and!pEF7,!respectively
,!which!were!derived!from!pJCP055
!
[
S
20]
.
!
pEF3!was!constructed!from!pJCP055!using
!
primers
!
PEF
143!and!144
!
to!amplify!the!
BRG1
!
exon
,!followed!by!digestion!with!XhoI!
and!ligation
.
!
Integration!was!checked!
with!PEF145,!ABO3,!PEF146,!and!ABO6.
!
pEF7!was!constructed!from!pJCP055!using!
primers!PEF161!and!162
!
to!amplify!the!
RIM101#
exon
,
!
followed!by!digestion!with!
XhoI!and!ligation.
.
!
Integration!was!checked!with!PEF96,!ABO3,!P
EF163,!and!ABO6.
!
!
Light!
m
icroscopy
!
!
!
15!μl!of!liquid!culture!from!the!suspension!co
O
culture!assay!was!visualized!on!an!
Axiovert!200M!microscope!(Carl!Zeiss,!Oberkochen,!Germany)!and!images!were!
acquired!with!Zeiss!Axiovision!Software,!version!4.!At!least!th
ree!technical!
replicates!and!two!biological!replicates!were!examined!for!each!sample.
!
!
qPCR
!
!
Quantitative!real!time!PCR!was!performed!as!described!
[
S
21]
.!Brie
fly,!cDNA!was!
synthesized!from!10!
μ
g!total!RNA!using!Superscript!II!Reverse!Transcriptase!
(
Invitrogen,!
18064
O
014),!according!to!the!manufacturer’s!instructions.!cDNA!was!
amplified!in!a!Bio
O
rad!C1000!Thermocycler!and!monitored!using!Sybr!Green!dye!
(Sigma!12
9K2163).!Fluorescence!was!measured!on!a!Bio
O
rad!CFX96!Real
O
Time!
System.!Data!were!analyzed!using!Bio
O
rad!CFX!Manager!software!version!2.0.!At!
least!2!biological!replicates!were!performed!for!each!condition.!Gene!expression!
was!normalized!against!either!
DYN
1#
or!
PAT1#
expression.
!
!
Quantification!of!
a
ggregation
!
!
To!quantify!aggregation
!
in!suspension!co
O
cultures
,!1!ml!
of!
culture!was!centrifuged!
for!30!seconds!at!500!rpm.!100!μl!supernatant!and!100!μl!pellet!were!
removed,!and!
OD
600!
was!read!using!the!Tecan
!
19!
Infinite!M1000!PRO!microplate!reader.!
At!least!
two!biological!replicates!and!two!technical!replicates!were!measured!for!each!
strain.
!
!
Supplemental
!
References
!
S
1.
!
Hernday,!A.!D.,!Lohse,!M.!B.,!Fordyce,!P.!M.,!No
bile,!C.!J.,!DeRisi,!J.!L.,!and!
Johnson,!A.!D.!(2013).!Structure!of!the!transcriptional!network!controlling!
white
O
opaque!switching!in!Candida!albicans.!Mol.!Microbiol.!
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–
35.
!
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Synnott,!J.!M.,!Guida,!A.,!Mulhern
O
Haughey,!S.,!Higgins,!D.!G.,!and!Butle
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Noble,!S.!M.,!and!Johnson,!A.!D.!(2005).!Strains!and!strategies!for!large
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–
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!
Banerjee,!M.,!Thompson,!D.!S.,!Lazzell,!A.,!Carlisle,!P.!L.,!Pierce,!C.,!Monteagudo,!
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O
Ribot,!J.!L.,!and!Kadosh,!D.!(2008).!UME6,!a!novel!filament
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–
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Tripathi,!G.,!Wiltshire,!C.,!Macaskill,!S.,!Tournu,!H.,!Budge,!S.,!and!Brown,!A.!J.!P.!
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O
ordinates!morphogenetic!and!metabolic!responses!to!amino!
acid!starvation!in!Candida!albi
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Pande,!K.,!Chen,!C.,!and!Noble,!S.!M.!(2013).!Passage!through!the!mammalian!
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–
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Nobile,!C.!J.,!Andes,!D.!R.,!Nett,!J.!E.,!Smith,!F.!J.,!Yue,!F.,!Phan,!Q.
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E.,!Filler,!S.!G.,!and!Mitchell,!A.!P.!(2006).!Critical!role!of!Bcr1
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Nobile,!C.!J.,!Schneider,!H.!a,!Nett,!J.!E.,!Sheppard,!D.!C.,!Filler,!S.!G.,!Andes,!D.!R.,!
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